Vector for anti-HPV vaccine and transformed microorganism by the vector

ABSTRACT

Expression vectors that can efficiently produce virion capsid protein, tumor-associated protein of human papillomavirus on a microbial surface. Bacterial strains harboring such surface display vectors, and the use of the bacterial strains or their extracts or purified products as complex vaccines, are also described. The surface display vectors contain one or more than two genes selected from among pgsB, pgsC and pgsA, encoding a poly-χ-glutamic acid synthetase complex (pgsBCA) of a  Bacillus  sp. strain, and genes that encode virion capsid proteins, tumor-associated proteins of human papillomavirus. Methods for preparing the foregoing vectors, vaccines and transformed microorganisms are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional filed under the provisions of 35 USC 120 of U.S.patent application Ser. No. 10/530,083 filed Apr. 1, 2005 as a U.S.national phase under 35 USC 371 of international patent applicationPCT/KR2003/002163 filed Oct. 17, 2003, which in turn claims priority ofKorean Patent Application No. 10-2002-0063378 filed Oct. 17, 2002.

TECHNICAL FIELD

The present invention relates to a vector for expressing a vaccineagainst a surface antigen of papilloma virus, a microbial transformantand a vaccine using said microbial transformant or purified extractthereof.

BACKGROUND

Cell surface display is new technique by which a desired protein isexpressed and attached onto a cell surface of microbe and is anapplication by using molecular biological information as the mechanismof protein secretion is elucidated. Precisely, the cell surface displayuses a microbial cell surface protein derived from bacteria, yeast orthe like as a surface anchoring motif to produce an exogenous protein ona cell surface. It has a broad range of application and thus, can beused to produce recombinant live vaccines, to prepare and screen apeptide/antigen library and to make whole cell absorbent, whole cellbioconversion catalyst and the like. Namely, this technique isconsiderably potential for an industrial use, since a variety ofexogenous protein expressed onto a cell surface decides the scope ofindustrial application.

Cell surface carrier is the most important factor to express anexogenous protein onto a cell surface successfully. To select aneffective anchoring motif expressing an exogenous protein onto a cellsurface is essential for this technique.

Accordingly, the surface expression carrier makes ready for severalfeatures as follows: Above all, some secretion signal is essential tohelp an exogenous protein pass through a cell inner membrane and finallyarrive at a cell surface. Second, a targeting signal is required toanchor an exogenous protein stably onto a cell outer surface. Third, ananchoring motif seldom affects a cell growth even if massively expressedonto a cell surface. Fourth, an exogenous protein sustains the3-dimensional structure to be expressed stably, regardless of a proteinsize. However, a surface expression carrier satisfying all the terms isnot found yet and is complemented only to settle some disadvantage ofabove cases.

In a broad sense, the surface anchoring carrier is now classified to 4kinds, including cell outer membrane protein, lipoprotein, secretoryprotein, surface organelle protein such as flagella protein. In case ofGram negative bacteria, a surface protein present on a cell outermembrane such as Lam B, Pho E (Charbit et al., J. Immunol., 139:1658-1664, 1987; Agterberg et al., Vaccine, 8: 85-91, 1990), Omp A andthe like is often exploited as a surface anchoring carrier. Besides, alipoprotein such as Tra T (Felici et al., J. Mol. Biol., 222: 301-310,1991), peptidoglycan associated lipoprotein (PAL) (Fuchs et al.,Bio/Technology, 9: 1369-1372, 1991), Lpp (Francisco et al., Proc. Natl.Acad. Sci. USA, 489: 2713-2717, 1992) and the like has been adopted andalso fimbriae protein such as Fim A or Fim H adhesin of type I fimbriae(Hedegaard et al., Gene, 85: 115-124, 1989) and pili protein such as PapA pilu subunit have been attempted to produce an exogenous protein.Furthermore, ice nucleation protein (Jung et al., Nat. Biotechnol, 16:576-560, 1998; Jung et al., Enzyme Microb. Technol, 22(5): 348-354,1998); Lee et al., Nat. Biotechnol., 18: 645-648, 2000), Klebsiellaoxytoca pullulanase (Komacker et al., Mol. Microl., 4: 1101-1109, 1990),Neisseria IgA protease (Klauser et al., EMBO L., 9: 1991-1999, 1990) orthe like have been reported to become a surface anchoring motif. In caseof Gram positive bacteria, it is known that a malaria antigen iseffectively produced by using protein A derived form Staphylococcusaureus as a surface anchoring motif and that a surface coat protein fromlactic acid bacteria is displayed well on a cell surface. Thus, thesurface protein of Gram positive bacteria is verified to become a cellsurface anchoring protein.

Previously, the present inventors have investigated a synthetic complexgene of poly-. γ-glutamate, pgs BCA derived from Bacillus sp. strain, asto whether it is applicable for new surface anchoring motif.Practically, pgs BCA gene was used to develop a novel recombinant vectorexpressing an exogenous protein onto a microbial surface and a methodfor producing an exogenous protein onto a cell surface in a large scale(Korean Patent Application No. 10-2001-48373).

Furthermore, the surface expression carriers described above have beentried a lot to produce various pathogenic antigens or antigenicdeterminants stably through the genetic engineering technique on ahighly productive bacterial surface. Especially, it is reported that anexogenous immunogen can induce more consistent and stronger immunereactions when expressed onto a non-pathogenic bacterial surface andorally administered as a live vaccine, compared with typical vaccinessuch as detoxified pathogenic bacteria or virus.

Since the microbial surface structure acts as an adjuvant enhancing theantigenicity of exogenous protein expressed onto a cell surface, theimmune reaction is known to be induced by live bacteria within a body.It is a remarkable event to develop a recombinant live vaccine ofnon-pathogenic bacterium through this surface expression system.

Human papilloma virus (hereinafter, referred to as “HPV”) is presumed toworld-widely infect more than 50% of all adult people. Especially, 4types of HPV including HPV 16, 18, 31 and 45 are confirmed to cause acervical cancer to more than 80% (Lowry D. R., Kirnbauer R., Schiller J.T., Proc. Natl. Acad. Sci., 91: 2436-2440, 1994). Papilloma virus ishighly species-specific and small DNA tumor virus and belongs to familyPapovaviridae which is infected to mammals such as human, cow, rabbit,sheep and so on and provokes a wart or papilloma on skin or mucosa(Pfister H., Adv. Cancer Res. 48: 113-147, 1987). Among these species,HPV are known to approximately 70 types and from more than 20 typescause tumors on skin mucosa of oral cavity or genital organs. Precisely,HPV 16 (type) and HPV 18 are reported to cause cervical cancer mostlycovering women cancers.

Cervical cancer is found frequently in women, next to breast cancerworld-widely. WHO (World Health Organization) has reported that cervicalcancer occurring newly is over 5 hundred thousand cases every year andmore than 3 hundred thousand of patients are died from cervical cancerevery year in the world. Especially in the developing country, cervicalcancer is a major cause of women death (Pisani P., Parkin D. M., FerlayJ., Int. J. Cancer 55: 891-903, 1993). IARC statistics showed that themost effective way to eradicate papilloma virus infection is toadminister a preventive vaccine for the future, since the developingcountry has even more chronic patients than the advanced country.

In order to develop certain vaccine against virus, the animal culturesystem should be equipped properly and exploited to produce and purifyvirus particles in a large scale. However, HPV is hardly changed to avirus particle in vitro or in vivo, since the virion is formed only in afully differentiated keratinocyte. Thus, there are severe problems todevelop vaccines against cervical cancer for the prevention and thetreatment as well as to produce virus enough for this researches. In abroad sense, 2 types of vaccine including prophylactic vaccine andtherapeutic vaccine are focused as a method for producing vaccinesagainst cervical cancer. For a purpose, the prophylactic vaccinegenerates a stronger neutralizing antibody by HPV L1/L2 antigen and thusprevents a host from HPV infection. Even if already infected, it makesthe disease no more progressed. In the meantime, the therapeutic vaccineuses HPV E6/E7, induces specific cellular immune reactions anddegenerates lesions or malignant tumor.

As searched out for last 20 years, HPV infected to human epithelium hasvarious kinds of genotype and is associated with several benign andmalignant tumors. Such an experimental data and discovery as to HPVpromotes to develop HPV vaccine. HPV recombinant virus like particle isassumed more optimistic among several HPV vaccine candidates sincebetter for an immune reaction than any other viruses except papillomavirus, through vaccine efficacy experiments in animal model and human(Koutsky L. A., Ault K. A., Wheeler C. M., Brown D. R., Barr E., AlvarezF. B., Chiacchierini L. M., Jansen K. U., N. Engi. J. of Med. 347(21):1645-1651, 2002). Furthermore, HPV infection is confirmed recently to bethe most essential cause of cancer definitely and scientists becomeinterested in HPV studies and directly participate in this so as toaccelerate the development of HPV vaccines globally. Nowadays, HPVvaccines widely known exploits HPV recombinant protein, HPV recombinantvirus like particle, HPV DNA and the like to manufacture products.

In bacteria, yeast, animal cell and so on, the recombinant proteinproduced from some HPV partial composition through the recombinant DNAtechnology and a synthetic peptide in which some major epitope issynthesized chemically are tried to develop vaccines. Generally,recombinant proteins are produced through a common system such asbacteria, yeast, baculovirus, recombinant vaccinia virus and the like,by which several researches are accomplished to produce HPV recombinantproteins and to identify the antibody forming ability against HPV inserum, the induction of cellular immune reaction and the like. However,the virus system using animal and insect cells is disadvantageous andthus contaminated during cultivation and hard to be purified. Includingthe synthetic peptide, overall cases cost high and are commerciallylimited in the industry, since papilloma virus infected patients areoften found in the developing countries.

Practically, HPV L1 virus like particle (hereinafter, referred to as“VLP”) has been disclosed to be produced as a live recombinant vacciniavirus through a mammary gland cell culture (Hagensee, M. E., Yaegashi,N., Gallowat, D. A., J. Virol. 67: 315-322, 1993) and VLP has beenreported to generate neutralizing antibodies in a mouse model system(Schiller J. T., Lowry, D. R., Seminars in Cancer Biol. 7: 373-382,1996). The therapeutic vaccine has been exploited by using HPV E6 and E7protein uniquely expressed in cervical cancer (Bubenik J., Neoplasma 49:285-289, 2002). In addition, HPV E6/E7 protein has been searched out asan immune target to treat cervical cancer and to develop a therapeuticvaccine, since it is a cancer specific antigen and associated with thecancerization of HPV infected cells. Actually, it is demonstrated thatwhen HPV E6/E7 protein produced by the microbial system is administeredto tumor cell-injected mice, the tumor formation is prevented anddelayed (Gao L., Chain B., Sinclair C., J. Gen. Virol. 75: 157-164,1994; Meneguzzi G., Cern C., Kieny M. P., Virology 181: 62-69, 1991). Asshown in other cases, live virus vaccine has also problems to provoke anviral proliferation excessively and is liable to stay in a researchlevel. Unfortunately, it takes a long time to be commercialized and alsoneeds considerable clinical trials. In order to overcome such adisadvantage, virus vectors which are inhibited or deficient in thereplication are explored but not commercialized yet (Moss B., Proc.Natl. Acad. Sci. USA 93: 11341-11348, 1996).

On the other hand, vaccine studies by using bacterial vectors areproceeded actively. It is disclosed that HPV 16 VLP produced fromattenuated Salmonella typhimurium inducibly generate antigen specificantibodies in mouse mucous membrane or whole body. Besides, vaccinecomposed of synthetic peptides uses only essential synthetic epitopeenough to induce an immune reaction for the vaccination andparticularly, an epitope inducing cytotoxic T lymphocyte (CTL) againstHPV 16 E6/E7 has been already elucidated (Ressing M. E., Sette A.,Brandt R. M., J. Immunol. 154: 5934-5943, 1995).

In addition to such a trials, vegetables including tomatoes, potatoesand so on are utilized to produce viral antigens in plants and also avegetable transformant itself is being attempted toward oral vaccine oredible vaccine. As a model case, hepatitis B surface antigen particle(Thavala Y. F. and C. J. Artzen. Proc. Natl. Acd. Sci. USA 92:3358-3361) and capsid protein L1 and L2 of papilloma virus (KoreanPatent Application 10-2000-0007022) are exemplified. In the plantsystem, however, HPV L1 protein is expressed in so small amount and sohard for the purification to limitedly engaged in commerce.

Therefore, since human papilloma virus is considered to be very ofteninfected to people in the developing countries, in order to prevent andeffectively treat tumors derived from papilloma virus onto skin mucus oforal cavity or genital organs, it is deeply required to develop a novelmethod for preparing human papilloma viral antigens more economicallyand stably.

DISCLOSURE OF THE INVENTION

In order to settle above-mentioned technical problems, the object of thepresent invention is to provide a recombinant vector and a microbialtransformant which can produce HPV antigen through a microbial surfaceexpression system.

In addition, another object of the present invention is to provide avaccine for treating and preventing mucosal tumor which is comprised ofa microbial transformant in which HPV antigen is expressed onto a cellsurface, a crude HPV antigen extracted from the transformant or a HPVantigen purified from the microbial transformant as an effectivecomponent.

In order to attain the above-mentioned object, the present inventionprovides a surface expression vector for preparing a vaccine whichcontains one or more than two genes encoding poly-.γ-glutamatesynthetase complex selected among pgs B, pgs C and pgs A and a surfaceantigen protein gene from human papilloma virus (HPV).

The gene pgs B, pgs C and pgs A mentioned in the present inventioninclude nucleotide sequences comprising SEQ ID NO. 1, SEQ ID NO. 2 andSEQ ID NO. 3 respectively.

The surface antigen protein gene of the present invention can be anykind of gene encoding a HPV surface component protein. For example, acapsid protein of human papilloma virus, HPV L1 or HPV L2 antigenprotein gene can be used solely or more than two genes, coordinately.The major capsid, HPV L1 antigen protein gene is preferable to be used.

The tumor-associated antigen gene of the present invention can be anykind of gene encoding a tumor-associated protein of HPV and atumor-associated antigen protein of human papilloma virus, HPV E6 or HPVE7 antigen protein gene can be used solely or more than two genes can beused coordinately. Besides, the modified tumor-associated gene E6 and E7can be used as an antigen and the major tumor-associated antigen proteinof HPV, E7 antigen protein is preferable to be used.

More preferably, the gene encoding poly-.γ-glutamate synthetase complexof the present invention can be pgs A.

Furthermore, the present invention relates to a microbial transformanttransformed with the recombinant vector for preparing a vaccinedescribed above.

Any kind of microorganism can be applied for the present invention, ifnon-toxic to a living body or attenuated. For example, Escherichia coli,Salmonella typhi, Salmonella typhimurium, Vibrio cholera, Mycobacteriumbovis, Shigella and the like can be selected as Gram negative bacteriumand Bacillus, Lactobacillus, Lactococcus, Staphylococcus, Lysteriamonocytogenesis, Streptococcus and the like as Gram positive bacteriumproperly.

The present invention provides various vaccines for treating andpreventing mucosal tumors which exploits microbial transformant itselfexpressing an antigen protein onto a cell surface, or uses crude extractof cell membrane components after microbes are disrupted, or exploits anantigen protein purified from a microbes as an effective component. Thatis to say, the vaccine of the present invention can be used for atherapeutic drug or a prophylactic drug for treating or preventing atumor which is caused by HPV and occurs on the mucosal membrane of oralcavity or genital organs, especially women cervical cancer.

The vaccine of the present invention can be administered orally or beedible, and injected hypodermically or peritoneally and can be a washingsolution for genital organs. When administered directly to women genitalorgan, a host cell can be preferably selected from useful bacteria suchas Lactobacillus sp. strain, which is clear to those skilled in thisart.

Besides, the vaccine of the present invention can be sprayed for thenasal cavity.

The mucus immunization is very important to prevent HPV infection sincethe HPV infection is often caused onto the mucosal surface. The oralvaccine using a microbial transformant itself is expected to be moreeffective to prevent HPV than the parental vaccine, since a microbeexpressing HPV antigen onto a cell surface is advantageous to induce amucosal response on the membrane more efficiently.

Concretely, the present invention provides the recombinant vector for avaccine use, pHCE2LB:pgsA-HPV L1 (See FIG. 1) which contains pgs A gene,a poly-.γ-glutamate synthetase complex gene derived from Bacillus sp.strain and expresses HPV L1 protein, a fused protein connecting theC-terminus of pgs A and the N-terminus of HPV L1 onto a cell surface ofGram negative bacterium or Gram positive bacterium and also themicrobial transformant for the surface expression. The Escherichia colitransformant transformed with the above-mentioned vector for a vaccineuse has been deposited separately (accession number: KCTC 10349 BP).

In addition, the present invention provides the surface expressionrecombinant vector for a vaccine use, pHCE2LB:pgsBCA-HPV E7 (See FIG. 5)which contains pgs BCA gene, a poly-.γ-glutamate synthetase complex genederived from Bacillus sp. strain and expresses HPV E7 protein, a fusedprotein connecting the C-terminus of pgs A and the N-erminus of HPV E7onto a cell surface of Gram negative bacterium or Gram positivebacterium and also the microbial transformant for the surfaceexpression. The Escherichia coli transformant transformed with theabove-mentioned vector for a vaccine use has been deposited separately(accession number: KCTC 10520 BP).

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which;

FIG. 1 depicts the genetic map of the recombinant vectorpHCE2LB:pgsA-HPV L1 which uses Gram negative bacterium and Gram positivebacterium as a host cell for the surface expression.

FIGS. 2A and 2B depicts the expression of HPV L1 antigen fused with pgsA within Salmonella strain and Lactobacillus strain transformed with therecombinant vector pHCE2LB:pgsA-HPV L1 for the surface expression.

FIG. 3A depicts the IgG antibody titer against HPV L1 antigen in serumfrom mouse in which Lactobacillus casei transformed with the recombinantvector pHCE2LB:pgsA-HPV L1 of the present invention and then verified toexpress the antigenic determinant onto a cell surface, is orallyadministered in a proper amount for some time.

FIG. 3B depicts the IgG antibody titer against HPV L1 antigen in mousewashing solution of intestine, bronchial tube, lung and vagina to whichLactobacillus casei transformed with the recombinant vectorpHCE2LB:pgsA-HPV L1 and then verified to express the antigenicdeterminant onto a cell surface, is orally administered in a properamount for some time.

FIG. 4 depicts the cytolysis activity of cytotoxic T lymphocyte in aspleen cell obtained from mouse in which Lactobacillus casei transformedwith the recombinant vector pHCE2LB:pgsA-HPV L1 and then verified toexpress the antigenic determinant onto a cell surface, is orallyadministered in a proper amount for some time.

FIG. 5 depicts the genetic map of the recombinant vectorpHCE2LB:pgsBCA-HPV E7 for the surface expression which uses Gramnegative bacterium and Gram positive bacterium as a host cell.

FIG. 6 depicts the expression of HPV E7 antigen fused with pgs A inLactobacillus strain which is transformed with the recombinant vectorpHCE2LB:pgsBCA-HPV E7 for the surface expression, by performing westernblotting with specific antibodies.

FIG. 7 depicts the proliferation rate of tumor cells obtained from mousein which Lactobacillus casei transformed with the recombinant vectorpHCE2LB:pgsBCA-HPV E7 and then verified to express the antigenicdeterminant onto a cell surface, is orally administered in a properamount for some time and challenged according to a time lapse.

FIG. 8 depicts the genetic maps of the cloning vector pGNBCA and therecombinant vector pGNBCA-HB168 for the surface expression as describedin Indirect Example of the present invention.

FIG. 9 depicts the surface expression of hepatitis B virus surfaceantigen protein from Gram negative bacterium transformed with therecombinant vector pGNBCA-HB168 by performing western blotting andfluorescent activating cell sorting flow cytometry as described inIndirect Example of the present invention.

FIG. 10 depicts the genetic maps of the cloning vector pGNCA and therecombinant vector pGNCA-HB168 for the surface expression as describedin Indirect Example of the present invention.

FIG. 11 depicts the surface expression of hepatitis B virus surfaceantigen protein from Gram negative bacterium transformed with therecombinant vectors (pGNCA-HB168:A2, pGNA-HB1 68:A3 and pGNHB-A:A4) byperforming western blotting and fluorescent activating cell sorting flowcytometry as described in Indirect Example of the present invention.

EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrated as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Especially, HPV type 16 L1 antigen protein gene is adopted in followingExamples, but any kind of antigen protein gene, a HPV capsid proteingene such as L1, L2 or so on from any other types of HPV strains can beused solely or more than two coordinately. In following Examples, HPVtype 16 E7 gene, a major antigen protein gene associated with a cancerinduction is used but any other cancer inducing antigen protein gene ofother HPV strains can be selected solely or coordinately.

Besides in following Examples, the cell outer membrane protein gene, pgsBCA participating in the poly-.γ-glutamate synthesis is collected fromBacillus subtilis var. chungkookjang (accession number: KCTC 0697 BP),but the vector including all the kinds of pgs BCA gene from Bacillus sp.strain producing poly-.γ-glutamate, microbial transformants using thevector or the like can be within the scope of the present invention.Precisely, other pgs BCA genes having more than 80% homology ofsequences present in the pgs BCA of Bacillis subtilis chungkookjang andderived from other microbial strains can be used to construct the vectorfor a vaccine use, which may be within the scope of the presentinvention.

As demonstrated in following Examples and Indirect Examples, pgs BCAgene can be utilized all or partially to construct the vector for avaccine use, which may be within the scope of the present invention.

Furthermore in following Examples, Salmonella typhi as Gram negativebacterium and Lactobacillus as Gram positive bacterium are used as ahost cell for the above-mentioned vector, but in addition to thesebacteria, other Gram positive or Gram negative bacteria can be used forthe same result, which is clear to those skilled in this art.

Also in following Examples, the microbial transformant itselftransformed with the vector for a vaccine use is only disclosed to applyfor a living body as a live vaccine. However, according to generalinformation in vaccine associated technical fields, it is natural toobtain the same or similar results even if crude extract from the abovemicrobial transformant or purified proteins are administered into aliving body.

Example 1 Construction of the Recombinant Vector pHCE2LB : pgsA: HPV L1for the Surface Expression

Cell outer membrane protein derived from Bacillus sp. strain andparticipating in the synthesis of poly-.γ-glutamate is exploited. Amongthe cell outer membrane gene, pgsBCA, gene pgsA was used to prepare therecombinant vector pHCE2LB:pgsA-HPV L1 which can express the majorcapsid protein L1 of human papilloma virus Type 16 (hereinafter,referred to as “HPV”) onto a cell surface by using Gram negativebacteria and Gram positive bacteria as a host cell.

Above all, the gene encoding HPV L1 is introduced into the surfaceexpression vector pGNA which uses Gram negative bacteria as a host cell(obtained from an applicant of Korean Patent Application No.10-2001-48373). Precisely, approximately 1.5 kb of human papilloma virusgene cloned in pUC19 was utilized as a template and oligonucleotidesencoding HPV L1 and containing the nucleotide sequence of SEQ ID. NO. 4or SEQ ID. NO. 5 as a template and then the polymerase chain reactionwas performed. As a result, 1518 bp-sized gene was amplified.

The primers of SEQ ID. NO. 4 and SEQ ID. NO. 5 were made to include therecognition site of restriction enzyme Bam HI and Hind III present inthe cloning vector pGNA for the surface expression. The HPA L1 antigengene amplified above was digested with the restriction enzyme Bam HI andHind III and ligated and adjusted in translation codons to theC-terminal region of cell outer membrane protein gene pgsA whichparticipates in the synthesis of poly-.γ-glutamate and is derived fromthe cloning vector pGNA so as to manufacture the recombinant vectorpGNA-HPV L1.

In order to obtain the DNA fragment containing HCE promoter, pgsA andHPV L1 from the recombinant vector prepared above, the recombinantvector pGNA-HPV L1 was digested with the restriction enzyme Nhe I andSca I and the resulting fragment was inserted to the restriction enzymeXba I and Sma I site within a multi-cloning site of common cloningvector pAT19 for Gram positive bacteria so as to construct therecombinant vector pHCE2LB:pgsA-HPV L1 (See FIG. 1).

The recombinant vector for the surface expression in the presentinvention was transformed to Escherichia coli and the bacterialtransformant including pHCE2LB:pgsA-HPV L1 was deposited on Oct. 4, 2002to Korea Research Institute of Bioscience and Biotechnology, Gene Bank(KCTC, 52 Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea) withthe accession number KCTC 10349 BP.

Example 2 Surface Expression of HPV L1 Fused with pgsA

Gram negative bacterium Salmonella typhi Ty21a was transformed with therecombinant vector pHCE21LB:pgsA-HPV L1 for the surface expressiondescribed above and examined the protein expression of HPV L1 antigenfused with pgsA in Salmonella typhi Ty21a. Then, Gram positive bacteriumLactobacillus was transformed, identified the presence of therecombinant vector pHCE21LB:pgsA-HPV L1 within Lactobacillus strain andexamined the protein expression of HPV L1 antigen fused with pgsA (SeeFIG. 2). The bacterial expression of HPV L1 antigen associated with theC-terminus of pgsA gene participating in the synthesis ofpoly-.γ-glutamate was examined by performing SDS-polyacrylamide gelelectrophoresis and western blotting with specific antibodies againstHPV L1.

Concretely, Salmonella typhi Ty21a transformed with the recombinantvector pHCE2LB:pgsA-HPV L1 was cultured onto 500 mL flask including 50mL of LB medium (yeast extract 5 g/L, tryptone 10 g/L, salt 5 g/L, pH7.0) containing 100 mg/L of antibiotic (erythromycin) and induced forthe surface expression. In addition, Lactobacillus casei was transformedwith the recombinant vector pHCE2LB:pgsA-HPV L1, cultured onto MRSmedium (Lactobacillus MRS, Becton Dickinson and Company Sparks, USA) at37.degree. C. in static culture, proliferated and induced for thesurface expression.

Afterward, Salmonella typhi Ty21a and Lactobacillus casei were inducedfor the surface expression and proteins were obtained in the same celldensity, and denatured for sample uses. Then the proteins were analyzedthrough SDS-polyacrylamide gel electrophoresis and in a fractionatedstate, transferred onto PVDF membrane (polyvinylidene-difluoridemembrane: Bio-Rad). The resulting PVDF membrane was submerged in ablocking buffer (50 mM Tris-HCL, 5% skim milk, pH 8.0), shaked for 1hour to be blocked, and reacted for 12 hours with a monoclonal primaryantibody against HPV L1 proteins which was derived from mouse anddiluted to 1,000 fold in the blocking buffer. The completely reactedmembrane was washed with the buffer and reacted for 4 hours with a mousesecondary antibody which was conjugated with biotins and diluted to1,000 fold in the blocking buffer. Then, the resulting membrane waswashed with the buffer, reacted with avidin-biotin reagent for 1 hourand washed again. The washed membrane became chromophored by addingH.sub.2O.sub.2 and DAB solution and was identified to have a specificbinding between specific antibody against HPV L1 and the above fusedprotein. (See FIG. 2)

In FIG. 2A, lane 1 is Salmonella typhi Ty21a, host cell not transformedand lane 2, 3 is Salmonella typhi Ty21a transformed with the recombinantvector pHCE2LB:pgsA-HPV L1. In FIG. 2B, lane 1 is Lactobacillus caseinot transformed and lane 2 is Lactobacillus casei transformed with therecombinant vector pHCE2LB:pgsA-HPV L1. As illustrated in Figs, thefused protein expressed from the recombinant vector pHCE21LB:pgsA-HPV L1was verified at 97.4 Kda band. Since pgsA has about 41.8 KDa and L1protein, about 55.6 Kda, the band having above 97.4 Kda is elucidated asa fused protein of pgsA and L1 protein.

Example 3 Induction of Immune Reaction in Lactobacillus Expressing HPVL1 Antigen Onto Cell Surface

The recombinant vector pHCE2LB:pgsA-HPV L1 for the surface expressionwas transformed to Gram positive bacterium, Lactobacillus casei, inducedto express the antigen onto cell surface of Lactobacillus casei asdescribed in Example 2 and then the cell outer membrane protein pgsAparticipating in the synthesis of poly-.γ-glutamate and the HPV 16 fusedwith L1 antigen were examined for the antigenicity.

Concretely, the recombinant vector pHCE2LB:pgsA-HPV L1 of the presentinvention was transformed to Lactobacillus casei for the surfaceexpression, harvested to adjust each cell to the same concentration andwashed several times with PBS buffer (pH 7.4). Then, Lactobacillus inwhich HPV16 L1 antigen is expressed onto cell surface and Lactobacillusnot transformed were administered in 5.times.10.sup.10 bacteria to anoral cavity of 4.about.6 week BALB/c mouse, three times every other dayin the first week and the second week and injected additionally after 2weeks, three times every other day. As a standard group, mice which wereadministered with HPV 16 L1 virus like particle (VLP) expressed fromyeast twice every other day were used.

In a 2-week interval after orally administered and venous injected, micewere victimized. Then, sera of each mouse group were collected tomeasure IgG antibody titers against HPV 16 L1 antigen within serum andsuspension solutions washing internal organs, bronchial tube, lung andvagina were tried to estimate IgA antibody titers against HPV 16 L1antigen by using ELISA method (Enzyme-linked immunosorbent assay) (SeeFIG. 3 a, 3 b). For the ELISA method calculating IgG and IgA antibodytiters against HPV 16 L1 antigen, HPV 16 L1 virus like particle (VLP)expressed from yeast was used as an antigen. Precisely, for measuringIgG antibody titer, horseradish peroxidase conjugated anti-mouse IgG andfor calculating IgG antibody titer, horseradish peroxidase conjugatedanti-mouse IgA were utilized.

In FIG. 3 a, ( ) is a group not transformed and orally administered withLactobacillus casei; ( ) a group intravenously injected with L1 viruslike particle of HPV 16 (VLP); and ( ) a group orally administered withLactobacillus casei transformed with the vector pHCE2LB:pgsA:HPV. InFIG. 3 b, is a group not transformed and orally administered withLactobacillus casei; a group intravenously injected with L1 virus likeparticle of HPV 16 (VLP); and a group orally administered withLactobacillus casei transformed with the vector pHCE2LB:pgsA:HPV.

Consequently as illustrated in FIG. 3 a, the serum solution which wasobtained from BALB/c mouse group administered with Lactobacillus caseitransformed with the vector pHCE2LB:pgsA-HPV L1 and diluted wasconfirmed to have a considerably higher IgG antibody titer against humanHPV L1 antigen than that of a standard BALB/c mouse group, and toincrease the antibody titers, which can be compared with those of agroup intravenously administered with HPV 16 L1 virus like particle(VLP).

As illustrated in FIG. 3 b, the suspension solution which washedinternal organs, bronchial tube, lung, the internal part of vagina orthe like of BALB/c mouse group administered with Lactobacillus caseitransformed with the vector pHCE2LB:pgsA-HPV L1 and diluted, wasconfirmed to increase IgG antibody titer against human HPV L1 antigen,which can be compared with those of the standard group intravenouslyadministered with HPV 16 L1 virus like particle (VLP). Especially, thesuspension of internal organ had the more remarkable IgA antibody titeragainst HPV 16 L1 antigen.

Accordingly, the microbial transformant of the present invention wasverified to produce IgG antibody against HPV as an index of whole bodyimmune induction and also IgA antibody against HPV as an index oflimited immune, mucose immune induction and thus, the microbialtransformant can be applied for a live vaccine.

Example 4 Cytolysis Activity of Spleen Cell from Experimental AnimalImmunized with Lactobacillus Expressing HPV L1 Antigen Onto Cell Surface

In order to investigate whether a cell-mediated immune reaction is alsoinduced among immune reactions through the same process, splenocyteswere separated from the experimental mice immunized with Lactobacillusexpressing HPV L1 antigen onto a cell surface and measured as to theactivity of cytotoxic lymphocyte (CTL).

Concretely, the splenocyte of Balb/c mouse not immunized and 10.mu.g ofsynthetic HPV 16 L1 peptide were incubated together at 37.degree. C. for3 hours and irradiated with 4,000 rad so as to prepare stimulatingcells.

As described in Example 3, the splenocytes of Balb/c mice immunized withLactobacillus expressing HPV L1 antigen onto a cell surface wereseparated, mixed with a stimulating cell in which a synthetic peptidewas loaded and cultivated for 6 days so as to prepare an effector cell.At this moment, the cell ratio of stimulating cell and effector cell wasadjusted to 2:1 and additionally stimulated in 6 days. Before one dayfor an assay, cells used for a target cell were incubated by adding10.mu.g of synthetic HPV 16 L1 peptide. For the day of assay, .sup.51CrO.sub.4 was added to peptide-loaded target cell in 100.mu.Ci/10.sup.6cell, incubated for 2 hours and washed. The effector cell prepared wasallotted to a 96 well plate in a ratio to 5,000 cells/well target cellfor the range of 1:100.about.1:25. The target cell and the effector cellwere mixed and incubated at 37.degree. C. for 4 hours. After 4 hours,supernatant was collected to measure the release of .sup.51CrO.sub.4.The maximum release was at the point that 1% Triton X-100 and targetcells were blended and the spontaneous release at the point that mediaand target cells were mixed and then the specific lysis was convertedaccording to a following formula.[Specific lysis=100*(experimental cpm−spontaneous cpm)/maximumcpm−spontaneous cpm]

The converted result of cytolysis activity was illustrated in FIG. 4.

Consequently as depicted in FIG. 4, the splenocyte of Balb/c mouse whichwas administered with Lactobacillus casei transformed with therecombinant vector pHCE2LB:pgsA-HPV L1 was identified to have a higherratio of specific cytolysis than that of Balb/c mouse which wasadministered with Lactobacillus not transformed.

Therefore, the immune reaction induced by Lactobacillus expressing HPV16 L1 antigen of the present invention was verified to be acell-mediated immune reaction which activates a cytotoxic lymphocyte asa major effect of an oral vaccine.

Example 5 Construction of the Recombinant Vector pHCE2 LB:pgsBCA:HPV E7for the Surface Expression

The cell outer membrane protein derived from Bacillus sp. strain andparticipating in the synthesis of poly-.γ-glutamate is exploited. Amongthe cell outer membrane gene, pgs BCA gene was used to prepare therecombinant vector pHCE2LB:pgsA-HPV-E7 which can express the majorantigen protein associated with a cancer induction E7 of HPV 16 onto acell surface by using Gram negative bacteria and Gram positive bacteriaas a host cell.

Above all, the gene encoding HPV 16 E7 is introduced into the surfaceexpression vector pGNBCA which uses Gram negative bacteria as a hostcell (obtained from an applicant of Korean Patent Application No.10-2001-48373). Precisely, approximately 324 bp of human papilloma virusgene cloned in pUC19 was utilized as a template and oligonucleotidesencoding HPV 16 E7 and containing the nucleotide sequence of SEQ ID. NO.6 or SEQ ID. NO. 7 as a template and then the polymerase chain reactionwas performed. As a result, 324 bp-sized gene was amplified.

The primers of SEQ ID. NO. 6 and SEQ ID. NO. 7 were made to include therecognition sites of restriction enzyme Bam Hl and HindIII present inthe cloning vector pGNBCA for the surface expression. The HPV E7 antigengene amplified above was digested with the restriction enzyme Bam HI andHindIII and ligated and adjusted in translation codons to the C-terminalregion of cell outer membrane protein gene pgsA which participates inthe synthesis of poly-.γ-glutamate and is derived from the cloningvector pGNBCA so as to manufacture the recombinant vector pGNBCA-HPV E7.

In order to obtain the DNA fragment containing HCE promoter, pgsBCA andHPV E7 from the recombinant vector pGNBCA-HPV E7 prepared above, therecombinant vector was digested with the restriction enzyme Nhe I andSca I and the resulting fragment was inserted to the restriction enzymeXbaI and Sma I site within the multi-cloning site of common cloningvector pAT19 for Gram positive bacteria so as to construct therecombinant vector pHCE2LB:pgsBCA-HPVE7 (See FIG. 5).

The recombinant vector for the surface expression in the presentinvention was transformed to Escherichia coli and the bacterialtransformant including pHCE2LB:pgsBCA-HPVE7 was deposited on Oct. 7,2003 to Korea Research Institute of Bioscience and Biotechnology GeneBank (KCTC, 52 Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea)with the accession number KCTC 10520 BP.

Example 6 Surface Expression of HPV E7 Fused with pgsA

Gram negative bacterium Lactobacillus was transformed with therecombinant vector pHCE21LB:pgsBCA-HPV E7 for the surface expressiondescribed above, identified the presence of the recombinant vectorpHCE21LB:pgsBCA-HPV E7 within Lactobacillus strain and examined theprotein expression of HPV E7 antigen fused with pgsA (See FIG. 6).

For this purpose, the surface expression vector was transformed toLactobacillus and induced through the same process described in Example2. Then, the expression of HPV E7 antigen protein fused with theC-terminus of pgsA gene participating in the synthesis ofpoly-.γ-glutamate was confirmed by performing SDS-polyacrylamide gelelectrophoresis and western blotting with specific antibodies againstpgsA (See FIG. 6A) and HPV E7 (See FIG. 6B) (See FIG. 6). In FIG. 6,lane 1 is Lactobacillus casei not transformed; and lane 2 and 3 areLactobacillus casei transformed with the recombinant vectorpHCE2LB:pgsBCA-HPV E7.

As illustrated in Figs., about 60.8 Kda band of fused protein wasverified from the recombinant vector pHCE2LB:pgsBCA-HPV E7. Since pgs Ahas about 41.8 Kda and HPV 16 E7 antigen protein has about 19 Kdatypically, the above 60.8 Kda band was elucidated to a fused protein ofpgs A and HPV 16 E7 antigen protein.

Example 7 Challenge of Tumor Cell to Experimental Animal Immunized withLactobacillus Expressing HPV E7 Antigen onto a Cell Surface

The recombinant vector pHCE21 LB:pgsBCA-HPV E7 for the surfaceexpression was transformed to Gram positive bacterium, Lactobacilluscasei, induced to express the above antigen onto the surface ofLactobacillus casei through the same process described in Example 2 andthen the inhibition effect upon the tumor proliferation was examined bythe immunization of HPV 16 E7 antigen fused with cell outer membraneprotein, pgs A participating in the poly-.γ-glutamate synthesis.

Concretely, Lactobacillus casei transformed with the recombinant vectorpHCE2LB:pgsBCA-HPV E7 for the surface expression and Lactobacillus nottransformed were treated as described in Example 3 and 5.times.10.sup.10transformants were orally administered to 4.about.6 week—C57/BL/6 mousethree times in the first week and the second week every other day andafter 2 weeks, additionally injected three times every other day.

After last injected, tumor cell line TC-1 (1.times.10.sup.4/50.mu.l)expressing HPV E7 was injected subcutaneously on the left side of mouseto be challenged.

Tumor cell line TC-1 expressing HPV E7 protein is induced from theprimary lung cell of C57/BL/6 mouse which is immunized and transformedwith HPV 16 E6 and E7 gene and activated human c-Ha-ras gene asdemonstrated in the prior art [Lin et al., Cancer Res. 56:21-26, 1996]

In 2 weeks after tumor challenged, the tumor size was measured. Threetimes a week every other day, the longest and the shortest length werecalculated and indicated to be doubled. The data measuring the tumorsize after tumor challenged were illustrated in FIG. 7.

As shown in FIG. 7, tumor cell challenged with C57/BL/6 mouse which wasadministered with Lactobacillus casei transformed with the vectorpHCE2LB:pgsBCA-E7 was remarkably inhibited to proliferate, compared withthan the result of C57/BL6 mouse administered with Lactobacillus nottransformed.

Therefore, it is confirmed that the immune reaction induced byLactobacillus in which HPV16 E7 antigen of the present invention wasexpressed onto a cell surface prevent tumor cells from theproliferation.

Indirect Example Construction of the Recombinant Vector for a VaccineCombining pgs B, pgs C and pgs A and Experiment for Expressing ExogenousProtein onto a Cell Surface by Using the System

It is confirmed that the surface expression vector can be constructed toinclude any one or more than two genes among pgs B, pgs C and pgs Aencoding poly-.γ-glutamate synthetase complex and gene encodingexogenous protein and transformed to microbes so as to be expressed ontothe microbial surface.

Accordingly, it is also identified indirectly that the vactor for avaccine use including any one or more than two genes among pgs B, pgs Cand pgs A encoding poly-.γ-glutamate synthetase complex and geneencoding HPV antigen protein of the present invention can bemanufactured.

In Indirect Example, the plasmid pGNBCA and pGNCA is the same with theplasmid vector pGNpgsBCA and pGNpgsCA of the present inventionrespectively.

Indirect Example 1 Construction of the Recombinant Vector pGNBCA-HB168and Surface Expression of Antigenic Determinant Forming NeutralizingAntibody of S Antigen

Cell outer membrane gene, pgs BCA participating in poly-.γ-glutamatesynthesis derived from Bacillus sp. strain was utilized to construct therecombinant vector pGNBCA-HB 168 expressing antigenic determinantforming neutralizing antibody of hepatitis B virus S antigen onto thecell surface by using Gram negative bacterium as a host cell.

In order to introduce hepatitis B virus S antigen gene to the surfaceexpression vector pGNBCA by using Gram negative bacterium as a hostcell, approximately 1.4 kb of hepatitis B virus gene cloned in thecommon cloning vector pUC8 was used as a template and oligonucleotidescontaining the nucleotide sequences of SEQ ID NO. 8 and SEQ ID NO. 9 asa primer. Then S antigen gene was amplified by performing the polymerasechain reaction. At this moment, the amplified gene is 168 bp in thesize.

The primers of SEQ ID NO. 8 and SEQ ID NO. 9 described above were madeto include the recognition site of restriction enzyme Bam HI and HindIII present in the surface expression vector pGNBCA. The hepatitis Bvirus S antigen gene amplified above was digested with the restrictionenzyme Bam HI and Hind III and ligated to the C-terminus of cell outermembrane gene participating in poly-.γ-glutamate synthesis, previouslyprepared and adjusting the translation codon. As a result, therecombinant vector pGNBCA-HB 168 was prepared as described above (SeeFIG. 8)

The recombinant vector pGNBCA-HB168 for the surface expression wasutilized to express the antigenic determinant forming neutralizingantibody of hepatitis B virus S antigen in Escherichia coli and toexamine the surface expression.

The surface expression vector constructed in Example 2 was transformedto Escherichia coli and cultivated in 50 mL of LB medium (yeast extract5 g/L, tryptone 10 g/L, salt 5 g/L, pH 7.0) containing 100 mg/Lantibiotic (ampicillin) and then induced for the surface expression.

The antigenic determinant forming neutralizing antibody of S antigenfused with the C-terminus of poly-.γ-glutamate synthetase gene wasexpressed in bacteria and identified by performing SDS-polyacrylamidegel electrophoresis and western blotting with specific antibodiesagainst S antigen. Concretely, proteins obtained in the same cellconcentration were denatured to prepare a sample and analyzed byperforming SDS-polyacrylamide gel electrophoresis and then, fractionatedproteins were transferred to PVDF membrane. The PVDF membrane completedto transfer proteins was shaked and blocked for 1 hour by using blockingbuffer (50 mM Tris HCI, 5% skin milk, pH 8.0). Afterward, the polyclonalprimary antibody against S antigen derived from sheep was diluted to1,000.times. in blocking buffer and reacted for 12 hours. The reactedmembrane was washed with buffer solution and incubated for 4 hours bydiluting the secondary antibody against sheep conjugated with biotin inblocking buffer to 1,000.times. The reacted membrane was washed withbuffer solution and incubated with avidin-biotin reagent for 1 hour andagain washed. Onto the washed membrane, H.sub.20.sub.2 and DAB solutionwere added as a substrate and coloring reagent and became colored so asto identify the specific binding in between specific antibody against Santigen and the above mentioned fused protein (See FIG. A). In FIG. 9,lane 1 is host cell JM109 not transformed and lane 2 is the transformantpGNBCA-HB 168/JM 109. As illustrated in Figs, approximately 48 KDa bandof fused protein was verified by the plasmid pGNBCA-HB 168.

In order to confirm the direct expression of the antigenic determinantforming neutralizing antibody of S antigen, Escherichia coli induced wasseparated respectively to soluble, inner membrane, outer membranethrough the outer membrane fractionation method and then, identified byperforming SDS-polyacrylamide gel electrophoresis and western blottingwith antibodies against S antigen. Concretely, Escherichia colitransformant induced to express the fused protein onto a cell surfaceand E. coli not transformed were harvested with adjusting to the samecell concentration, washed in buffer solution (10 mM HEPES buffer, pH7.4) several times, suspended in buffer solution containing 10.mu.g/mllysozyme, 1 mM PMSF and 1 mM EDTA and reacted for 4.about.10 minutes.Afterward, DNase (0.5 mg/.mu.l) and RNase (0.5 mg/ml) were added andcells were sonicated, separated to E. coli and cellular debris bycentrifuging at 10,000.times.g for 4, 20 minutes and collected thefraction including periplasm and cytoplasm of E. coli. Then, thecollected pellet was suspended in PBS buffer (pH 7.4) containing 1%Sarcosyl (N-lauryl sarcosinate, sodium salt), separated to innermembrane protein of E. coli in a supernatant and to outer membraneprotein in a cell pellet by centrifuging at 15,000.times.g for 4, 2hours and thus the fractions were obtained. Each fraction was identifiedby performing polyacrylamide gel electorphoresis and western blottingwith antibodies against S antigen and from E. coli fractions theantigenic determinant forming neutralizing antibodies of S antigen wasverified to exist in the outer membrane (See FIG. 9A; the result ofwestern blot in E. coli membrane fraction). In FIG. 9, lane 1 is JM 109strain not transformed; lane 2, whole cell of the transformantpGNBCA-HB168/JM 109; lane 3, soluble fraction of the transformantpGNBCA-HB168/JM109; lane 4, the outer membrane fraction of thetransformant pGNBCA-HB168/JM109; and lane 5, the outer membrane fractionof the transformant pGNBCA-HB168/JM109.

It is confirmed that the antigenic determinant forming neutralizingantibody of S antigen was expressed onto E. coli cell surface on accountof the C-terminus of poly-.γ-glutamate synthetase protein, through thefluorescence activating cell sorting (FACS) flow cytometry. For animmunofluorescence staining, E. coli induced was harvested to adjust thecell concentration to the same level, washed with PBS buffer (pH 7.4)several times, suspended in 1 ml buffer containing 1% bovine serumalbumin, and reacted with polyclonal primary antibody against S antigenderived from sheep after diluted to 1,000.times. for 4.about.12 hours.The cells completed to react were washed several times in buffersolution, suspended in 1 ml buffer containing 1% bovine serum albuminand then reacted for 3.about.4 hours after diluting thebiotin-associated secondary antibody. Again, the reacted cell was washedseveral times with buffer solution, suspended in 0.1 ml buffercontaining 1% bovine serum albumin and then reacted with biotin-specificstreptoavidin-R-phycoerythr- in, coloring reagent after diluted to1,000-fold.

The reacted E. coli was washed several times and examined through thefluorescence-activating cell sorting flow cytometry. As a result, theantigenic determinant protein forming neutralizing antibody of S antigenwas identified to be expressed onto a cell surface, compared with E.coli not transformed (See FIG. 9B). In FIG. 9B, white color is derivedfrom JM 109 not transformed and black color, from the transformantpGNBCA-HB168/JM109. As illustrated in FIG. 9, the antigenic determinantforming neutralizing antibody of S antigen was not expressed in E. colinot transformed, but in E. coli transformant transformed with thesurface expression vector, the antigenic determinant formingneutralizing antibody of S antigen was clearly verified.

Indirect Example 2 Construction of the Recombinant Vector pGNCA-HB168and Surface Expression of the Antigenic Determinant Forming NeutralizingAntibody of Hepatitis B Virus S Antigen

Cell outer membrane gene, pgs C and pgs A among pgs BCA participating inpoly-.γ-glutamate synthesis derived from Bacillus sp. strain wasutilized to construct the recombinant vector for the surface expressionby using Gram negative bacterium as a host cell.

In order to obtain the gene encoding the N-termini and the C-termini ofpgs C and pgs A from cell outer membrane protein participating inpoly-.γ-glutamate synthetase, whole chromosome was used as a templateand oligonucleotides containing the nucleotide sequences of SEQ ID NO.10 and SEQ ID NO. 11 as a primer. Then the polymerase chain reaction wasperformed.

The primer corresponding to the N-terminus was made to contain therecognition site of restriction enzyme Nde I present in the expressionvector pHCE 19T(II). At this moment, the amplified region hasapproximately 1.6 kb size from the N-terminus of pgs C to the C-terminusof pgs A which is a cell outer membrane gene participating inpoly-.γ-glutamate synthesis.

The gene amplified by the polymerase chain reaction was digested withthe restriction enzyme Nde I and Bam HI and inserted to theconstitutively high expression vector pHCE19T(II) already digested withNde I and Bam HI. As a result, the new vector having about 5.3 kb sizewas to prepared and named to pGNCA in which the cell outer membraneprotein gene participating in the poly-.γ-glutamate synthesis do nothave the translation termination codon and new recognition sites ofrestriction enzymes are added.

The recombinant vector pGNCA-HB168 expressing the antigenic determinantforming neutralizing antibody of hepatitis B virus S antigen onto thecell surface was constructed by using the cell outer membrane gene, pgsC and pgs A among pgs BCA participating in poly-.γ-glutamate synthesisand Gram negative bacterium as a host cell as described in Example 2.The recombinant vector pGNCA-HB 168 prepared above was illustrated inFIG. 10.

The recombinant vector pGNCA-HB168 was utilized to produce the antigenicdeterminant forming neutralizing antibody of hepatitis B virus S antigenin E. coli and examined the surface expression.

For this purpose, the recombinant vector was transformed to E. coli andinduced to be expressed through the same process described in Example 3,and then, the antigenic determinant forming neutralizing antibody ofhepatitis B virus S antigen fused with the cell outer membrane proteinpgs CA was identified to be expressed by performing SDS polyacrylamidegel electrophoresis and western blotting with antibodies against Santigen.

In FIG. 11, lane 1 is host cell JM 109 not transformed; lane 2 thetransformed pGNCA-HB168/JM109. As illustrated in FIG. 11, approximately48 KDa band of fused protein from the plasmid pGNCA-HB168 was verified.

INDUSTRIAL APPLICABILITY

The present inventors have developed a method for efficiently expressingcapsid L1 protein of human papilloma virus onto a microbial surface byusing poly-.γy-glutamate synthetase gene, pgs BCA derived from Bacillussp. strain, especially onto a microbial surface of Lactobacillus orallyadministered and of Salmonella as a vaccine strain. Besides, therecombinant strain can be applied for a therapeutic and a prophylacticvaccine and the like. Since in case of HPV, the antibodies is notinductively produced in a large scale, the recombinant strain of thepresent invention expressing HPV antigen can be an economical vaccinesuch as an oral vaccine or directly applicable to vaginal legion, afterproliferated in a large scale economically and chiefly.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention.

Those skilled in the art will also appreciate that such equivalentembodiments do not depart from the spirit and scope of the invention asset forth in the appended claims.

1. A vector useful for preparing a vaccine, said vector including (i)one or more than two genes selected from among pgs B, pgs C and pgs Aencoding poly-γ- glutamate synthetase complex and (ii) an antigenprotein gene of human papilloma virus, in which said antigen proteingene is one or more than two genes selected from a group comprisingcapsid HPV L1 and HPV L2 of human papilloma virus.
 2. The vector ofclaim 1, which includes a pgs A gene encoding said poly-γ-glutamatesynthetase complex.
 3. The vector of claim 1, which is pHCE2LB:pgsA-HPVL1.
 4. A vaccine for treating or preventing mucosal tumors, comprisingan active ingredient including at least one of: (a) a recombinantmicroorganism transformed with a vector including (i) one or than twogenes selected from among pgs B, pgs C and pgs A encodingpoly-γ-glutamate synthetase complex and (ii) an antigen protein gene ofhuman papilloma virus, in which said antigen protein gene is one or morethan two genes selected from a group comprising capsid HPV L1 and HPV L2of human papilloma virus; (b) an extract from said recombinantmicroorganism; and (c) proteins purified from said recombinantmicroorganism.
 5. The vaccine of claim 4, which is adapted to beadministered orally or be edible.
 6. The vaccine of claim 4, which isadapted to be injected subcutaneously or peritoneally.
 7. The vaccine ofclaim 4, which is adapted to be sprayed to the nasal cavity.
 8. Avaccine of claim 5, which comprises a recombinant microorganismtransformed with the vector pHCE2LB: pgsA-HPV L1.
 9. The vaccine ofclaim 8, which is adapted to be administered orally or be edible. 10.The vaccine of claim 8, which is adapted to be injected subcutaneouslyor peritoneally.
 11. The vaccine of claim 8, which is adapted to besprayed to the nasal cavity.
 12. A washing solution for a genital organ,which includes as an effective component, a recombinant microorganismtransformed with the vector pHCE2LB: pgsA-HPV L1.
 13. A recombinantbacterium, which is selected from the group consisting of: (a) arecombinant Gram negative bacterium transformed with a vector including(i) one or more than two gene selected from among pgs B, pgs C and pgs Aencoding poly-γ-glutamate synthetase complex and (ii) an antigen proteingene of human papilloma virus, in which said antigen protein gene is oneor more than two genes selected from a group comprising capsid HPVL1 andHPVL2 of human papilloma virus; (b) a recombinant bacterium transformedwith the vector pHCE2LB: pgsA-HPV L1; and (c) an a recombinantEscherichia coli transformed with the vector KCTC 10349 BP.
 14. Therecombinant bacterium of claim 13, wherein said bacterium is a Gramnegative bacterium selected from among Escherichia coli, Salmonellatyphi, Salmonella typimurium, Mycobacterium bovis, and Shigella.
 15. Therecombinant bacterium of claim 13, which is transformed with the vectorpHCE2LB: pgsA-HPV L1.
 16. The recombinant bacterium of claim 13, whereinsaid bacterium is Salmonella.
 17. The recombinant bacterium of claim 13,which is a recombinant Escherichia coli transformed with the vector KCTC10349 BP.